1. Field of the Invention
This invention relates to a method for providing permanent, optically clear preparations of biological specimens on microscope slides for cytologic, histologic and pathologic study.
2. Description of the Prior Art
It is common practice in the medical field to provide thin sections of biological specimens on glass slides for microscopic study, such sections have been stained (to enhance definition for examination), dehydrated and subjected to a mounting process. Generally, such mounting process encloses each individual specimen in a medium supported on a microscope slide and wherein it is preserved indefinitely. The present trend is to use a solvent solution of hard synthetic resins as the mounting medium, or mountant. Such resins are chosen so that they (1) do not cause the stains with which the specimens are treated to fade, (2) yellow minimally with age, and (3) have refractive indices after drying which closesly approximate the average refractive index of tissues (1.530-1.570). If a mountant having a refractive index close to that of the specimen is used, an almost perfect transparency can be achieved. A .mu.-pinene resin is often used as mountant. For a listing of other available mountants, reference is made to table 5-1 on page 98 of Histopathic Technique and Practical Histo-chemistry by R. D. Lillie, McGraw-Hill Book Company, New York ( 1965).
Generally, the mountant is dissolved in a quantity of solvent, to provide proper viscosity for the convenient dispensing of a controlled volume thereof over the specimen. To provide a planar upper surface, necessary for proper microscopic examination, it is common practice to apply a glass cover slip over the dispensed mountant while still in liquid form. To remove all solvent, the presence of which can interfere with the examination process, the slides are subjected to an extended drying process, which may require several days or more. During such process, the slides are placed in a horizontal position, and sometimes within a warm environment, to facilitate solvent evaporation. Such solvent evaporation is essential to (1) harden the mountant, (2) seal the cover slip, so as to permit handling of the slides, and (3) avoid optical interference by the low refractive index of residual solvent during examination.
At the present time, the mounting and drying processes are done manually in the laboratory. Commonly, after removing the slide from the so-called clearing solution and quickly draining the same, one to several drops of mountant are deposited by the technician over the specimen. The quantity of such mountant should be sufficient to just occupy the space between the cover slip, when applied, and the specimen. Immediately, the cover slip is carefully positioned by the technician by being lowered gently upon the mountant. The technique contemplates that one side of the cover slip is placed on the mountant, which is then released to push before it a portion of the mountant as it descends into proper position. Often, the cover slip requires some adjustment by the technician, so as to achieve a thin even film of mountant, to eliminate any entrapped air bubbles from beneath the cover slip, and to properly center the cover slip over the specimen. Any surplus of mountant expressed beyond the cover slip should be quickly and carefully removed, for example, with absorbent paper, by the technician.
The most common difficulties encountered in the mounting of specimens are (1) a delayed application of the mountant after the specimen is removed from the clearing agent allows the drying of the specimen, whereby some deterioration of the tissue structure results, (2) the amount of mountant is inappropriate, whereby the resultant film beneath the cover slip is too thick or too thin or the plane of the cover slip is not parallel with that of the microscope slide, and (3) too rapid or careless application of the cover slip traps air bubbles, which become increasingly more difficult to release as the mountant begins to dry.
The prior art procedures, which have been only briefly described, have well-recognized disadvantages, among which are:
1. Since the specimen can be damaged if allowed to dry, the mountant must be carefully and quickly applied by the technician. PA1 2. Since the solvent in the mountant has a low index of refraction and diffuses only slowly out from under the cover slip during the drying process, the mountant is generally prepared with a minimum of solvent, usually sufficient only to allow convenient dispensing. However, if the placement of the cover slip is delayed, the surface of the mountant can begin to dry, which tends to increase the probability of trapping air bubbles under the cover slip. Accordingly, the cover slip must be both carefully and quickly positioned on the mountant. PA1 3. If an excess of mountant is dispensed, such excess must be expressed beyond the edges of the cover slip. Adequate removal of such excess may require the use of an appropriate solvent, which might flow under the cover slip and remove underlying mountant, ultimately leaving air spaces which would interfere with the examination. PA1 4. During the drying process, the slide must be set aside on a flat horizontal surface for a considerable period. Tilting of the slide during such process often causes the cover slip to slide off the tissue section. Also, since the edges of the mountant extend slightly beyond the periphery of the cover slip and diffusion of solvent from under such cover slip keeps such edges sticky, the slides cannot be stacked for efficient storage until the drying process is complete. PA1 5. Even after a prolonged drying process, a minimum quantity of solvent remains under the central portion of the cover slip. Inasmuch as the solvent has a relatively low refractive index, the optical properties of the slide remain sub-optimal for a long time period. PA1 6. If the tissue specimen should have a high point, a thick layer of mounting medium would be present under the cover slip. Accordingly, the drying process would be delayed and considerable shrinkage of the mounting medium would result as the solvent evaporates. Such shrinkage often occurs unevenly and locally, such as to create an air space under the cover slip which would obscure tissue detail.